Identification of a receptor binding region on the beta subunit of human follicle-stimulating hormone

Mouse epidermal growth factor (mEGF) and the beta subunit of follicle-stimulating hormone (hFSH) (hFSH-beta) have been shown to inhibit binding of intact hFSH to its testes membrane receptor in vitro. Both hFSH-beta and mEGF contain the tetrapeptide sequence Thr-Arg-Asp-Leu (TRDL). Previous results demonstrated that synthetic TRDL inhibited binding of intact hFSH to receptor. We therefore investigated the possibility that TRDL was located on an exposed region of FSH-beta using a polyclonal antiserum to hFSH [NHPP anti-hFSH batch 4 (AB4)] which recognized determinants on intact hFSH and its beta subunit, but not the alpha subunit. Pituitary FSH preparations from several mammalian species produced parallel inhibition curves in a heterologous [AB4 and 125I-labeled ovine FSH (125I-oFSH)] radioimmunoassay with relative potencies similar to those observed for the same preparations assayed by radioligand receptor assay. This antiserum also competitively inhibited 125I-FSH binding to receptor. Thus, AB4 appeared to recognize antigenic determinants that are highly conserved and located at or near regions involved with hormone recognition of receptor for FSH. Synthetic TRDL inhibited 50% of 125I-hFSH binding to antiserum at a concentration of 1.36 mg/tube (9 x 10(-3) M). Other tetrapeptides (Thr-Pro-Arg-Lys and Lys-Thr-Cys-Thr) had no inhibitory activity at comparable concentrations. A mixture of the free amino acids T, R, D, and L inhibited radioligand binding only at significantly higher concentrations than TRDL.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of cyanoketone on follicle-stimulating hormone (FSH) induction of receptors for FSH in granulosa cells of the rat

Follicle-stimulating hormone (FSH) enhances the conversion of testosterone or androstenedione into estradiol by stimulating the aromatase enzyme system. Estradiol also enhances FSH action. Thus, a synergistic action of FSH and estradiol may be required for maturation of ovarian follicles. We hypothesized that estradiol may be required for FSH action. Thus, blocking estrogen synthesis should prevent FSH-induced increases in FSH receptors. Hypophysectomized rats were divided into five groups and injected subcutaneously with: 1) saline, 2) cyanoketone (0.05 mg, blocks the conversion of pregnenolone to progesterone), 3) ovine FSH (oFSH, 200 micrograms), 4) cyanoketone then oFSH 24 h later, or 5) cyanoketone plus estradiol [or progesterone, testosterone, promegestrone (R5020), dihydrotestosterone (DHT), 2 mg], then FSH 24 h later. Animals were decapitated at 0, 12 or 24 h after an injection of oFSH, and membrane receptors for FSH and luteinizing hormone (LH), plus nuclear receptors for estradiol from granulosa cells, were measured. LH receptor levels were increased only after administration of FSH and estradiol. At 0 and 24 h, numbers of FSH or estradiol receptors were similar in saline- and cyanoketone-treated animals. FSH alone increased (P less than 0.01) FSH and estradiol receptors 3-fold and 4-fold, respectively, over controls by 12 and 24 h. Cyanoketone prevented these increases in FSH and estradiol receptors. Estradiol replacement fully reversed the effects of cyanoketone on FSH action. Replacement with progesterone and testosterone was able to only partially restore levels of FSH receptors; however, estradiol receptor numbers were also increased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of a follicle-stimulating hormone receptor-binding region in hFSH-beta-(81-95) using synthetic peptides.

Pituitary and placental glycoprotein hormones are heterodimers with alpha-subunits of identical primary structure, but dissimilar beta-subunits. Regions of structural similarity between the beta-subunits might be involved in interaction with the homologous alpha-subunits, and regions of structural dissimilarity could, therefore, be candidates for receptor interactions. A restrained matrix dot-plot analysis identified hFSH-beta-(8-32) and hFSH-beta-(55-65) as candidates for interaction with alpha-subunit. Therefore, by subtraction, hFSH-beta-(33-54) and hFSH-beta-(66-111) seemed candidates for regions of interaction with receptor. In a previous report we demonstrated that hFSH-beta-(33-53) represented a receptor-binding region of hFSH-beta. Analysis of structural parameters (flexibility, surface probability, secondary structure prediction, etc.) indicates similarities between hFSH-beta-(33-53) and hFSH-beta-(85-95), suggesting the latter might be the component of hFSH-beta-(61-111) interacting with the receptor. Testing of 11 synthetic peptides, corresponding to the primary structure of hFSH-beta, demonstrated that hFSH-beta-(31-45)-peptide amide, were unique in ability to inhibit 125I-follicle-stimulating hormone binding to receptor. hFSH-beta-(81-95)-peptide amide also stimulated estradiol biosynthesis in Sertoli cell cultures. The correlation between binding inhibition and surface probability, flexibility, and predicted secondary structure (alpha, extended, and turn) was highly significant (R2 = 0.87, p less than 0.0001). Regression significance for these parameters, taken individually, were very poor. Receptor-binding regions, therefore, appear to be characterized by a particular and complex arrangement of secondary structure motifs, surface probability, and flexibility.     

Chemical synthesis of peptide fragments of the hormone-specific beta-subunit of human follicle-stimulating hormone

In order to determine the specific antigenic determinants of human follicle-stimulating hormone (hFSH), hFSH-beta peptides with amino acid residues 33-49 (V2), 95-118 (V3), 76-118 (V3 + 1/2 C2), 1-33 (V1 + C1), 22-33 (1/2C1), and 95-107 (V3 + 1/4C2) according to the nomenclature of Stewart and Stewart [Stewart, M., & Stewart, F. (1977) J. Mol. Biol. 116, 175] as well as additional peptides with the residues 93-107, 91-107, 89-107, 87-107, and 85-107 were chemically synthesized. The peptides were examined in radioimmunoassay systems of FSH, luteinizing hormone (LH), or human chorionic gonadotropin (hCG). V3 + 1/2C2 and V1 + C1 showed immunological activity, whereas the other peptides did not. Antibodies were raised in rabbits against these peptides and examined for specific binding with hFSH, LH, thyroid-stimulating hormone (TSH), and hCG. V3 + 1/2C2 as well as V1 + C1 produced antisera, which specifically bound hFSH, hLH, and hTSH, indicating that the amino acid sequences contained in hFSH-beta peptides V3 + 1/2C2 and V1 + C1 share common antigenic sites with hLH and hTSH. Antisera were produced in rabbits against hFSH-beta, against reduced and S-aminoethylated hFSH-beta (AE-FSH-beta), and against AE-FSH-beta coupled to hemocyanin. Reduced and S-aminoethylated beta-subunit of FSH-beta coupled with hemocyanin produced antisera in rabbits that specifically bound only hFSH and not hLH, hTSH, or hCG.     

Sulfhydryl groups are involved in the interaction of FSH with its receptor

FSH has recently been reported to possess thioredoxin-like activity, presumably explained by the homology between a region of FSH-beta subunit and the active site of thioredoxin. The homologous sequence lies within a receptor binding region, which suggests a possible role for sulfhydryl groups in the formation of an active hormone-receptor complex and subsequent signal transduction. In order to determine the relevance of sulfhydryl groups on FSH-receptor interaction, we studied the effect of N-ethylmaleimide (NEM) and glutathione on FSH binding. The results indicate that free sulfhydryl groups, probably derived from the FSH receptor, are involved in ligand-receptor interaction.     

Cysteine residues in a synthetic peptide corresponding to human follicle-stimulating hormone beta-subunit receptor-binding domain 81-95 [hFSH-beta-(81-95)] modulate the in vivo effects of hFSH-beta-(81-95) on the mouse estrous cycle.

We have previously reported that synthetic peptide amides corresponding to subdomains of the human FSH 3-subunit, hFSH-beta-(33--53) and hFSH-beta-(81--95), interact with the external domain of the FSH receptor in two in vitro model systems. Consistent with these in vitro observations, we found that intraperitoneal (i.p.) administration of each of these peptides prolonged vaginal estrus in normally cycling mice in vivo. Both hFSH-beta-(33--53) and hFSH-beta-(81--95) contain cysteine (Cys) residues with free sulfhydryl groups of potential significance in receptor interactions. To assess the possible involvement of these groups in the in vivo effects of hFSH-beta-(33--53) and hFSH-beta-(81--95), synthetic peptide analogs were prepared in which all Cys residues were replaced with serine (Ser). In the present study, we demonstrate that the in vivo effect of hFSH-beta-(33--53) on the mouse estrous cycle, extension of vaginal estrus, was not changed by substitution of Cys-51 with Ser. In contrast, mice receiving the Ser-substituted analog of hFSH-beta-(81--95) had normal estrus stages, but were arrested in diestrus. hFSH-beta-(33--53)-(81--95), a linear peptide encompassing both domains, also prolonged vaginal estrus. The Ser-substituted analog of this peptide, however, prolonged vaginal estrus in some of the mice tested and induced cycle arrest at diestrus in others. hFSH-beta-(90--95), the active subdomain at the C-terminus of hFSH-beta-(81--95), extended vaginal estrus, but diestrus stages were of normal duration. Its Ser-substituted analog, however, prolonged the estrus stage of the majority of mice treated, but induced diestrus arrest in some. The differing responses to these peptides are presumably due to interactions of the synthetic peptides with different regions of the FSH receptor. This further suggests that one consequence of ligand interaction with multiple receptor binding domains may be variable effects on ovarian function, and that Cys to Ser analogs may have value in the design of a novel class of synthetic peptides capable of fertility regulation and control.     

Two receptor binding regions of human FSH show sense-antisense similarity to the human FSH receptor

The sequences of two receptor binding regions of the beta-subunit of the human follicle-stimulating hormone (hFSH-beta) were compared with the DNA-derived antisense peptide sequence of the hFSH receptor. A striking sense-antisense similarity was established between these receptor binding regions and the hFSH receptor. Based on this sense-antisense similarity four putative hormone binding regions on the N-terminal extracellular region of the hFSH receptor are identified.     

On the mechanism of action of lead in the testis: in vitro suppression of FSH receptors, cyclic AMP and steroidogenesis

Previous evidence has shown that prenatal and neonatal exposure to low levels of Pb result in decreased FSH binding and steroidogenesis in the testes at the onset of puberty. The purpose of the present study was to determine by in vitro methods, if Pb acts by interfering directly with hormone binding, cyclic AMP production and steroidogenic enzyme activity. Sertoli cells were isolated from testes of prepubertal rats and cultured in the presence of 2.64 x 10(-4)M of either NaAc (control) or PbAc for 1, 4, 24, 48, 96 or 144 hr. There was no reduction in FSH binding and in FSH-induced cyclic AMP after a 1-4 hr exposure to Pb. After a 24-hr exposure to Pb, the cells exhibited a 10-20% decrease in FSH binding and cyclic AMP production and after 96 hr there was a 75% decrease in these 2 parameters. The inhibition was greater in cells from 16 day old than from 20 day old rats, so that in the former, after a 144 hr exposure the FSH-induced cyclic AMP of the Pb exposed cells was only 3% of the amount produced by the NaAc exposed cells (i.e. a 97% inhibition). After in vitro exposure to Pb for 48 hr, the steroidogenic activity (progesterone conversion to steroid metabolites) of Sertoli cells was significantly reduced and their steroidogenesis was no longer stimulated by FSH. A crude testicular enzyme preparation containing 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) exhibited approximately 25% reduction in activity if the assay buffer contained PbCl2 instead of the equivalent in NaCl. Prolonged in vivo exposure to Pb resulted in approximately 50% reduction in 3 beta-HSD activity. This is the first indication that in the testis Pb may act directly (immediate effect) by suppressing enzyme activities, and indirectly (long term effect) by reducing gonadotropin-receptor binding and the resultant cyclic AMP production.     

FSH, the neglected sibling: evidence for its role in regulation of spermatogenesis and Leydig cell function

The role of follicle stimulating harmone(FSH) in male reproductive function remains a matter of debate although recent evidences strongly suggest a role despite the controversies that arose following the results obtained with FSH-beta null mice and observations from human FSH receptor mutations. This review summarizes the recent developments of our understanding on the role of FSH in male reproduction. Specifically the results obtained with FSH-beta and FORKO null mice are be discussed in light of our observations employing active and passive neutralization of endogenous FSH in rodents and primates along with other studies. On the basis of results obtained employing a variety of models it can be conclude unequivocally that FSH regulates Leydig cell function and is essential for normal quantitative spermatogenesis.     

Allosteric activation of the follicle-stimulating hormone (FSH) receptor by selective, nonpeptide agonists

The pituitary glycoprotein hormones, luteinizing hormone and follicle-stimulating hormone (FSH), act through their cognate receptors to initiate a series of coordinated physiological events that results in germ cell maturation. Given the importance of FSH in regulating folliculogenesis and fertility, the development of FSH mimetics has been sought to treat infertility. Currently, purified and recombinant human FSH are the only FSH receptor (FSH-R) agonists available for infertility treatment. By screening unbiased combinatorial chemistry libraries, using a cAMP-responsive luciferase reporter assay, we discovered thiazolidinone agonists (EC50's = 20 microm) of the human FSH-R. Subsequent analog library screening and parallel synthesis optimization resulted in the identification of a potent agonist (EC50 = 2 nm) with full efficacy compared with FSH that was FSH-R-selective and -dependent. The compound mediated progesterone production in Y1 cells transfected with the human FSH-R (EC50 = 980 nm) and estradiol production from primary rat ovarian granulosa cells (EC50 = 10.5 nm). This and related compounds did not compete with FSH for binding to the FSH-R. Use of human FSH/thyroid-stimulating hormone (TSH) receptor chimeras suggested a novel mechanism for receptor activation through a binding site independent of the natural hormone binding site. This study is the first report of a high affinity small molecule agonist that activates a glycoprotein hormone receptor through an allosteric mechanism. The small molecule FSH receptor agonists described here could lead to an oral alternative to the current parenteral FSH treatments used clinically to induce ovarian stimulation for both in vivo and in vitro fertilization therapy.     

Role of ERK1/2 in the differential synthesis of progesterone and estradiol by granulosa cells.

A major concept in mammalian ovarian physiology is that follicle-stimulating hormone (FSH) activates the granulosa cells (GCs) in the Graafian follicle to selectively produce estradiol, but not progesterone, during the follicular phase of the menstrual or estrous cycle. However, given the fact that FSH can induce production of both estradiol and progesterone by GCs cultured in vitro, it has been postulated for a long time that there is a factor present in the ovary that selectively prevents FSH-induced progesterone production. Here, we provide evidence that two members of the mitogen-activated protein kinase family, extracellular signal-regulated kinase-1 and -2 (ERK1/2) can differentially regulate FSH-stimulated estradiol and progesterone production. Using primary rat GCs from early antral follicles cultured in serum-free medium for 48 h, we found that the addition of a specific inhibitor of ERK1/2 activation, U0126, caused the attenuation or enhancement of FSH-induced progesterone or estradiol production, respectively, in a dose-dependent manner. Throughout the 48-h culture period in this culture system ERK1/2 molecules in their activated state (phospho-ERK1/2) were clearly detectable in GCs exposed to FSH. The addition of U0126 caused a decrease in the levels of phosphorylated but not unphosphorylated ERK1/2 which was maintained throughout the 48-h culture, suggesting that U0126 was continuously active to inhibit the phosphorylation of ERK1/2. The divergent regulation of FSH-induced progesterone and estradiol synthesis by U0126 was further supported by demonstrating that U0126 inhibits and stimulates FSH-induced mRNA levels of steroidogenic acute regulatory protein and P450 aromatase, respectively. Collectively, this study clearly identified ERK1/2 as the first intracellular signaling molecules that differentially regulate FSH-induced progesterone and estradiol synthesis in GCs.     

Involvement of transforming growth factor alpha in the regulation of rat ovarian X-linked inhibitor of apoptosis protein expression and follicular growth by follicle-stimulating hormone

The expression of X-linked inhibitor of apoptosis protein (XIAP), a member of a family of intracellular antiapoptotic proteins, is induced by FSH during follicular development in vivo. Whether the XIAP up-regulation by FSH (100 ng/ml) is a direct action of the gonadotropin and is important in the control of granulosa cell proliferation during follicular growth is unclear. The overall objective of the present study was to examine whether the FSH-induced XIAP expression and granulosa cell proliferation during follicular development is mediated by the secretion and action of intraovarian transforming growth factor alpha (TGFalpha). In rat follicles cultured for 2 and 4 days, FSH stimulated estradiol production, TGFalpha secretion, XIAP expression, and follicular growth. The theca cells are the primary follicular source of FSH-induced TGFalpha, as indicated by in situ hybridization. Intrafollicular injection of a neutralizing anti-TGFalpha antibody (50-200 ng/ml; immunoglobulin G as control) or addition of estradiol-antagonist ICI 182780 (0.5-100 nM) to the culture media suppressed FSH-induced XIAP expression and follicular growth. The effect of ICI 182780 could be partially reversed by high concentrations of estrogen (250 and 500 nM). Whereas TGFalpha (10-20 ng/ml) significantly increased granulosa cell XIAP content and proliferation in primary granulosa cell cultures, FSH alone was ineffective in eliciting the mitogenic response. Our results support the hypothesis that FSH stimulates granulosa cell proliferation via theca TGFalpha secretion and action in response to increased granulosa cell estradiol synthesis, and that XIAP up-regulation in response to FSH suppresses granulosa cell apoptosis and facilitates FSH-induced follicular growth.     

Involvement of G protein-coupled receptor kinases and arrestins in desensitization to follicle-stimulating hormone action.

FSH rapidly desensitizes the FSH-receptor (FSH-R) upon binding. Very little information is available concerning the regulatory proteins involved in this process. In the present study, we investigated whether G protein-coupled receptor kinases (GRKs) and arrestins have a role in FSH-R desensitization, using a mouse Ltk 7/12 cell line stably overexpressing the rat FSH-R as a model. We found that these cells, which express GRK2, GRK3, GRK5, and GRK6 as well as beta-arrestins 1 and 2 as detected by RT-PCR and by Western blotting, were rapidly desensitized in the presence of FSH. Overexpression of GRKs and/or beta-arrestins in Ltk 7/12 cells allowed us to demonstrate 1) that GRK2, -3, -5, -6a, and -6b inhibit the FSH-R-mediated signaling (from 71% to 96% of maximal inhibition depending on the kinase, P < 0.001); 2) that beta-arrestins 1 or 2 also decrease the FSH action when overexpressed (80% of maximal inhibition, P < 0.01) whereas dominant negative beta-arrestin 2 [319-418] potentiates it 8-fold (P < 0.001); 3) that beta-arrestins and GRKs (except GRK6a) exert additive inhibition on FSH-induced response; and 4) that FSH-R desensitization depends upon the endogenous expression of GRKs, since there is potentiation of the FSH response (2- to 3-fold, P < 0.05) with antisenses cDNAs for GRK2, -5, and -6, but not GRK3. Our results show that the desensitization of the FSH-induced response involves the GRK/arrestin system.     

转铁蛋白抑制大鼠卵泡颗粒细胞FSH受体的结合与维持

转铁蛋白能抑制大鼠卵泡颗粒细胞的分化,但其机理尚不清楚。本实验用已烯雌酚处理后分离的大鼠颗粒细胞,观察了转铁蛋白对FSH结合到颗粒细胞,以及在培养过程中对颗粒细胞FSH受体量维持的影响。结果表明,生理浓度范围的转铁蛋白部分地阻断了~(125)I-rFSH结合到颗粒细胞上;将转铁蛋白与FSH一起处理细胞,发现它能剂量依赖性地抑制FSH对颗粒细胞FSH受体的维持作用,并表现为孕酮及雌二醇的分泌减少。     

Hormone-induced desensitization of cultured rat granulosa cells to FSH

Monolayers of granulosa cells (GC) derived from immature hypophysectomized diethylstilbestrol-treated rats became refractory in terms of FSH-stimulable cyclic AMP production following exposure to the homologous hormone. In the presence of ovine FSH (5 μg/ml), maximal refractoriness was attained after 4 h of incubation. Upon removal of the FSH from the medium, the cells regained their full responsiveness within 24 h. The extent of desensitization was dependent upon the dose of FSH, and could not be overcome by increasing the dose of the hormone during the challenge period. Exposure of GC monolayers for 2–4 h to the protein synthesis inhibitors actinomycin D (8 μg/ml) and cycloheximide (5 μg/ml) on their own enhanced FSH-stimulable cyclic AMP production. When added together with FSH, the inhibitors did not prevent the process of desensitization to the hormone. The results suggest that the initial phases of FSH-induced desensitization do not require de novo protein synthesis.     

Regulation of cyclic adenosine 3',5'-monophosphate-dependent protein kinase activity and regulatory subunit RII beta content by basic fibroblast growth factor (bFGF) during granulosa cell differentiation: possible implication of protein kinase C in bFGF action.

We have previously shown that basic fibroblast growth factor (bFGF) inhibits the FSH-induced differentiation of cultured rat granulosa cells, as manifested by prominent reduction of the LH receptor expression. We now investigate the possible sites and mechanism of action of bFGF. Whereas bFGF decreased the cAMP formation induced by FSH, it enhanced the cAMP production caused by cholera toxin and forskolin, suggesting that bFGF exerted its inhibitory action on cell differentiation at a step to cAMP production. Photoaffinity labeling with 8-azido-[32P]cAMP revealed that bFGF markedly reduced the FSH-induced increase in the level of regulatory subunit RII beta of the cAMP-dependent protein kinase (PKA) type II. In contrast to its striking effect on RII beta expression (70-80% inhibition), bFGF decreased PKA enzymatic activity by only 30%. On the other hand, transforming growth factor-beta (TGF beta) slightly amplified the stimulatory action of FSH and antagonized the bFGF inhibitory effect on both LH receptor expression and RII beta synthesis. We report that the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol-13-acetate (TPA), which impaired granulosa cell differentiation, also abolished the RII beta synthesis induced by FSH. The activation of PKC by bFGF in granulosa cells was supported by the following findings: (i) bFGF markedly enhanced the production of diacylglycerol (2.3-fold stimulation at 5 min), the intracellular activator of PKC; (ii) bFGF promoted tight association of PKC to cellular membranes, a process that is believed to correlate with the enzyme activation; (iii) bFGF induced the phosphorylation of an endogenous M(r) 78,000/pI 4.7 protein that appears as a specific PKC substrate; (iv) bFGF mimicked the TPA-induced transmodulation of the epidermal growth factor (EGF) receptor, reducing by 36% the 125I-EGF binding on granulosa cells. We conclude that bFGF may exert its repressive action on RII beta synthesis, PKA activity, and granulosa cell differentiation by primarily targeting PKC activation.     

The follicle-stimulating hormone (FSH) receptor in testis: interaction with FSH, mechanism of signal transduction, and properties of the purified receptor

The follicle-stimulating hormone (FSH) receptor purified from calf bovine testis membranes appears to be an oligomeric glycoprotein, consisting of 4 disulfide-linked monomers of molecular weight about 60,000 each. Polyclonal antibodies to the hormone binding sites of the receptor have been developed. FSH interaction with the receptor seems to involve multiple discrete binding regions, which include amino acids 34-37 and 49-52 of the human FSH beta subunit. The interaction between FSH and the membrane-bound receptor is reversible at low temperatures but becomes increasingly irreversible as the temperature increases. FSH interaction with the soluble receptor is reversible over a wider temperature range. The hydrophobic effect is a significant factor in the initial hormone receptor interaction in each system. FSH bound to membrane receptors on cultured immature rat Sertoli cells is internalized and degraded to the level of amino acids. Current evidence suggests that the membrane receptor may exist as free receptor, and complexed with G-protein. A functional receptor/G-protein/adenylate cyclase complex has been reconstituted in liposomes. The G-protein of testis membranes contains both high and low affinity guanosine triphosphate (GTP) binding sites. Both are capable of modulating FSH receptor binding, whereas only the high affinity sites seem to be required for activation of adenylate cyclase. Although testis membranes contain a phosphatidylinositide hydrolysis system, the latter is not directly influenced by FSH.     

Progesterone and regulation of the follicle-stimulating hormone (FSH-beta) gene.

Progesterone (P) biphasically modulates follicle-stimulating hormone (FSH) secretion in the rat both in vivo and in vitro with the duration of estrogen priming determining the biphasic nature of the P action, probably through estrogen up-regulation of the anterior pituitary progesterone receptor (PR) levels. P has been also shown to regulate anterior pituitary levels of FSH-beta mRNA in the rat. Although the mechanism of this action has not been determined, steroids may regulate gene expression through the binding of liganded receptors to gene sequences known as hormone response elements (HRE); however, it is not known whether HRE's exist on the rat FSH-beta gene. We have localized a series of progesterone response elements (PRE)-like sequences on the rat FSH-beta gene and have begun testing the hypothesis that P modulates the expression of the rat FSH-beta gene through the direct binding of the P/PR complex to these PRE-like sequences. Electromobility shift assays indicate that these PRE-like sequences bind PR with high affinity and specificity. In addition, when a 361-base pair sequence, which contains the three PRE-like sequences localized in the upstream region of the gene, was cloned into a luciferase expression vector driven by a heterologous promoter and transiently transfected into anterior pituitary cell cultures, progestin stimulation elicited increased luciferase expression. These results indicated that the 361-base pair sequence conferred P-responsiveness to a heterologous promoter. The data further suggest that FSH synthesis in the rat is modulated by direct binding of PR to PRE-like sequences.     

L-cystine inhibits aspartate-beta-semialdehyde dehydrogenase by covalently binding to the essential 135Cys of the enzyme

Aspartate-beta-semialdehyde dehydrogenase (ASADH) from Escherichia coli is inhibited by L- and D-cystine, and by other cystine derivatives. Enzyme inhibition is quantitatively reversed by addition of dithiothreitol (DTT), dithioerythrytol, beta-mercaptoethanol, di-mercaptopropanol or glutathione to the cystine-inactivated enzyme. Cystine labeling of the enzyme is a pH dependent process and is optimal at pH values ranging from 7.0 to 7.5. Both the cysteine incorporation profile and the inactivation curve of the enzyme as a function of pH suggest that a group(s) with pK(a) of 8.5 could be involved in cystine binding. Stoichiometry of the inactivation reaction indicates that one cysteine residue from the enzyme subunit is reactive against cystine, as found by direct incorporation of radioactive cystine into the enzyme and by free-thiol titration of the enzyme with 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) before and after the cystine treatment. One mole of cysteine is released from each mol of cystine after reaction with the enzyme. ASA, NADP and NADPH did not prevent cystine inhibition. The [35S]cysteine-labelled enzyme can be visualized after electrophoresis in polyacrylamide gels and further detection by autoradiography. After pepsin treatment of the [35S]cysteine-inactivated enzyme, a main radioactive peptide was isolated by HPLC. The amino acid sequence of this peptide was determined as FVGGN(Cys)(2)TVSL, thus demonstrating that the essential 135Cys is the amino acid residue modified by the treatment with cystine.